[Histonet] double immunostaining on same species [Scanned]

Tue Jul 15 08:32:59 CDT 2008

There are several option for doing double or triple colocalisations using the same species of primary antibodies on any species.

Option 1

Combine a sensitive detection system e.g tyramide with a less sensitive detection system e.g indirect (using fluorescently labeled secondary). The theory here is that you can use your first primary Ab using a tyramide detection at a dilution beyond the detection limits of the second detection indirect). A normal serum block between the detections can prove effective.

Option 2

Use monovalent secondary antibodies for your detection e.g Goat anti Mouse Fab Biotinylated followed by a fluorescent streptavidin. Basically you repeat this detection sequentially for each primary using a different fluorophore. It can help to do a unconjugated fab block in between detections. In our hand this works best with monoclonal primaries but can have success with polyclonals as well but it is a bit trickier. Rule here is to use the same monovalent secondary for each detection. Because of the monovalent nature of the secondary there is little/no cross reaction. Unfortunately the monovalent secondaries on the market are polyclonal there for it is best to use the same secondary reagent as a different (polyclonal monovalent secondary) may recognize different epitopes on your primary.

Option 3

Use a tyramide detection for each of your primaries and perform a heat induced antigen retrieval step in between. This is pretty bomb proof. It can be used for double any species!!!!!!!!! providing your epitope is not destroyed by the HIER, in our hands generally HIER will improve antigenicity rather than be detrimental although this is not always the case, you just have to try it and see. To our knowledge this will only work with tyramide detections as the HIER seems to strip away any tissue bound antibodies, hence why it gives very clean co-localisations with no possibility of cross reactions. The HRP catalysed deposition of fluorescently labeled tyramide around the site of the HRP means that the fluorescent tyramide binds to the tissue around the site of the HRP labeled secondary and is not attached to the secondary antibody. For some reason the HIER does not affect the tyramide tissue binding, which is fortunate but it does destroy/denatured/strip away the antibodies and any possibility of cross reactions associated with them. Of course if one of your antibodies is destroyed by retrieval then do this detection first without retrieval! If retrieval destroys both your epitopes you are really unlucky and I wouldn't waste your money on a lottery ticket this week. 

Option 4

It does not have to be fluorescent, colourometric (DAB and fast blue e.g) can give pretty good results especially if the epitopes are not colocalised to the same cellular structure so nuclear and cytoplasmic or different cell types within the same tissue give good results. The colours do get a bit muddy if things colocalise to the same structure though. Again do a HIER step between detections to remove any possibility of cross reactions............sorted!

Finally if you are being daring and imaginative you can try a combination of the above methods BUT you must do appropriate controls to prove that you truly are seeing a real colocalisation as opposed to a detection cross reaction. Id love to take credit for thinking up these cunning ideas however this is no the case and a search of the literature should pull up the relevant publications.

Mike Millar, Sheila MacPherson, Arantza Esnal and Nancy Evans
MRC Human Reproductive Sciences Unit, Edinburgh, Scotland UK

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of histonet-request <@t> lists.utsouthwestern.edu
Sent: 14 July 2008 18:26
To: histonet <@t> lists.utsouthwestern.edu
Subject: Histonet Digest, Vol 56, Issue 17 [Scanned]

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Today's Topics:

   1. Reticular stain (Dunfee, LuWanda R)
   2. Re: Reticular stain (Rene J Buesa)
   3. Reply double immunostaining on same species, mouse (Gayle Callis)
   4. Re: Double immunostaining with two same source 1st Antibody (tf)
   5. Re: Reply double immunostaining on same species, mouse (tf)
   6. Re: Re: [Histonet] Double immunostaining with two same source
      1stAntibody (tf)
   7. Re: Re: RE: [Histonet] Anterograde tracing (tf)
   8. RE: freezing artefact holes on edge of cortex in mouse	brain
      sections (Aine Behan)
   9. Re: RE: freezing artefact holes on edge of cortex in
      mousebrain sections (tf)
  10. Re: RE: [Histonet] Double immunostaining with two same source
      1st	Antibody (tf)
  11. Schiff's removal (Blazek, Linda)
  12. NBT/BCIP staining for Alkaline Phosphatase
      (eplurbus <@t> u.washington.edu)

----------------------------------------------------------------------

Message: 1
Date: Sun, 13 Jul 2008 13:00:39 -0700
From: "Dunfee, LuWanda R" <ldunfee <@t> seattlecca.org>
Subject: [Histonet] Reticular stain
To: <histonet <@t> lists.utsouthwestern.edu>
Message-ID:
	<mailman.0.1216054800.32549.histonet <@t> lists.utsouthwestern.edu>
Content-Type: text/plain;	charset="us-ascii"

Hello,
I am working on the reticular stain that we are currently using.  We are
using a modified version of the Gordon and Sweet's stain.  It is not
selective enough, and it is picking up other collagen fibers.  I was
wondering if anyone had any suggestions.
Thank You,
LuWanda 
Seattle Cancer Care Alliance

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Notice of Privacy Practices, visit our web site at www.seattlecca.org. 

------------------------------

Message: 2
Date: Sun, 13 Jul 2008 13:35:37 -0700 (PDT)
From: Rene J Buesa <rjbuesa <@t> yahoo.com>
Subject: Re: [Histonet] Reticular stain
To: histonet <@t> lists.utsouthwestern.edu,	"Dunfee, LuWanda R"
	<ldunfee <@t> seattlecca.org>
Message-ID: <48288.32832.qm <@t> web65702.mail.ac4.yahoo.com>
Content-Type: text/plain; charset=iso-8859-1

Try Gomori's method.
René J.

--- On Sun, 7/13/08, Dunfee, LuWanda R <ldunfee <@t> seattlecca.org> wrote:

From: Dunfee, LuWanda R <ldunfee <@t> seattlecca.org>
Subject: [Histonet] Reticular stain
To: histonet <@t> lists.utsouthwestern.edu
Date: Sunday, July 13, 2008, 4:00 PM

Hello,
I am working on the reticular stain that we are currently using.  We are
using a modified version of the Gordon and Sweet's stain.  It is not
selective enough, and it is picking up other collagen fibers.  I was
wondering if anyone had any suggestions.
Thank You,
LuWanda 
Seattle Cancer Care Alliance

This electronic message transmission contains information which may be 
confidential or privileged. The information is intended to be for the 
use of the individual or entity named above. If you are not the 
intended recipient, be aware that any disclosure, copying, distribution 
or use of the contents of this information is prohibited. If you have 
received this electronic transmission in error, please leave a message 
via telephone at (206) 288-6266, notify me by electronic reply, and 
delete this message. Opinions and ideas in this message that do not 
relate to official business are understood as neither given nor 
endorsed by the Seattle Cancer Care Alliance. To view our complete 
Notice of Privacy Practices, visit our web site at www.seattlecca.org. 

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Message: 3
Date: Sun, 13 Jul 2008 17:01:21 -0600
From: "Gayle Callis" <gayle.callis <@t> bresnan.net>
Subject: [Histonet] Reply double immunostaining on same species, mouse
To: "Histonet" <histonet <@t> lists.utsouthwestern.edu>
Message-ID: <000e01c8e53c$5dfc8d90$6501a8c0 <@t> DHXTS541>
Content-Type: text/plain;	charset="iso-8859-1"

We do double and triple immunfluorescence staining on mouse tissues with all antibodies raised in same host species only on mouse tissues,  rat antiMouse.   It takes some careful blocking and organization, but it works beautifully.  Please contact me and I will discuss the details on how to set up the staining, but supply some details on what you want to see as green or red fluorescence.  This is only done on frozen sections and NOT on formalin fixed paraffin embedded tissue to eliminate autofluorescence. There are several ways to set this up, and we do use some biotinylated primary antibodies along with Molecular Probes Streptavidin Alexa fluor dyes.  Gayle M. CallisHTL/HT/MT(ASCP)Bozeman MT You can still have issues with the second red detection trying to stick to the first AB unless you do some very aggressive blocking.
Patsy

-----Original Message-----
Sent: Saturday, July 12, 2008 9:58 AM
To: histonet
Subject: [Histonet] Double immunostaining with two same source 1st Antibody

Hi all

Just wonder any one of you tried to perform IHC using two primary antibodies from same source, mice, for example?

The process is that 
Mice anti-A (1st IHC)->secondary antibody labeled with GREEN
fluroscence->wash
-> Mice anti-B (2nd IHC)-> Red fluroscence

then there's no cross reaction.

thx.

2008-07-12 

tf 
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Message: 4
Date: Mon, 14 Jul 2008 10:02:23 +0800
From: "tf" <tifei <@t> foxmail.com>
Subject: Re: [Histonet] Double immunostaining with two same source 1st
	Antibody
To: "Neil Fournier" <nfournier <@t> sasktel.net>,	"histonet"
	<histonet <@t> lists.utsouthwestern.edu>
Message-ID: <200807141002185117220 <@t> foxmail.com>
Content-Type: text/plain;	charset="gb2312"

Some one suggested a "citrate buffer" treatment before 2nd IHC.
However he als suggested be careful about losing anti-gen in this step.

Actually I used this step for BrdU staining pretreatment before adding HCl to broken DNA helix (brdu can be taken by cell and synthsized into DNA).
It seems that citrate buffer has more function than simply the antigen retrieval.

Can anyone comment on this further?

2008-07-14 

tf 

·¢¼þÈË£º Neil Fournier 
·¢ËÍÊ±¼ä£º 2008-07-14  03:26:20 
ÊÕ¼þÈË£º tifei <@t> foxmail.com 
³ËÍ£º 
Ö÷Ìâ£º [Histonet] Double immunostaining with two same source 1st Antibody 

I am extremely interested in the suggestions you receive. I would like to do a similar procedure. If possible, would you be able forward them to me.

Much appreciated, 

Neil

Neil M. Fournier
Department of Psychology
University of Saskatchewan
9 Campus Drive
Saskatoon, SK, Canada
S7N 5A5

Phone:  306-966-4024
    Fax:  306-966-6630 

------------------------------

Message: 5
Date: Mon, 14 Jul 2008 10:06:00 +0800
From: "tf" <tifei <@t> foxmail.com>
Subject: Re: [Histonet] Reply double immunostaining on same species,
	mouse
To: "Gayle Callis" <gayle.callis <@t> bresnan.net>,	"Histonet"
	<histonet <@t> lists.utsouthwestern.edu>
Message-ID: <200807141005554294023 <@t> foxmail.com>
Content-Type: text/plain;	charset="gb2312"

That's cool and please kindly let me know the detail.

Yes we used PFA to fix tissue and we have both microtome / cryostat to make sections.
the cryostat sections are embeded with O.C.T.
We have several mouse monoclonal antibodies, and I will use only Alexa fluroscence conjugated 2nd antibody rather DAB staining.

Thanks a lot.

2008-07-14 

tf 

·¢¼þÈË£º Gayle Callis 
·¢ËÍÊ±¼ä£º 2008-07-14  07:05:57 
ÊÕ¼þÈË£º Histonet 
³ËÍ£º 
Ö÷Ìâ£º [Histonet] Reply double immunostaining on same species, mouse 

We do double and triple immunfluorescence staining on mouse tissues with all antibodies raised in same host species only on mouse tissues,  rat antiMouse.   It takes some careful blocking and organization, but it works beautifully.  Please contact me and I will discuss the details on how to set up the staining, but supply some details on what you want to see as green or red fluorescence.  This is only done on frozen sections and NOT on formalin fixed paraffin embedded tissue to eliminate autofluorescence. There are several ways to set this up, and we do use some biotinylated primary antibodies along with Molecular Probes Streptavidin Alexa fluor dyes.  Gayle M. CallisHTL/HT/MT(ASCP)Bozeman MT You can still have issues with the second red detection trying to stick to the first AB unless you do some very aggressive blocking.
Patsy
-----Original Message-----
Sent: Saturday, July 12, 2008 9:58 AM
To: histonet
Subject: [Histonet] Double immunostaining with two same source 1st Antibody
Hi all
Just wonder any one of you tried to perform IHC using two primary antibodies from same source, mice, for example?
The process is that 
Mice anti-A (1st IHC)->secondary antibody labeled with GREEN
fluroscence->wash
-> Mice anti-B (2nd IHC)-> Red fluroscence
then there's no cross reaction.
thx.
2008-07-12 
tf 
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Message: 6
Date: Mon, 14 Jul 2008 10:08:38 +0800
From: "tf" <tifei <@t> foxmail.com>
Subject: Re: Re: [Histonet] Double immunostaining with two same source
	1stAntibody
To: "Igor Nasonkin" <nasonkini <@t> mail.nih.gov>,	"histonet"
	<histonet <@t> lists.utsouthwestern.edu>
Message-ID: <200807141008330513746 <@t> foxmail.com>
Content-Type: text/plain;	charset="gb2312"

Thanks very much Igor.

I'd like to share the information to other people who are interested in this.

"www.jacksonimmuno.com 
Check out their web site-technical pages-staining with 2 Abs from same
sources. Their protocols are good and they do work
Luck
igor"

2008-07-14 

tf 

·¢¼þÈË£º Igor Nasonkin 
·¢ËÍÊ±¼ä£º 2008-07-14  10:06:55 
ÊÕ¼þÈË£º tifei <@t> foxmail.com 
³ËÍ£º 
Ö÷Ìâ£º Re: [Histonet] Double immunostaining with two same source 1stAntibody 

Www.jacksonimmuno.com
Check out their web site-technical pages-staining with 2 Abs from same
sources. Their protocols are good and they do work
Luck
igor
On 7/13/08 10:02 PM, "tf" <tifei <@t> foxmail.com> wrote:
> Some one suggested a "citrate buffer" treatment before 2nd IHC.
> However he als suggested be careful about losing anti-gen in this step.
> 
> Actually I used this step for BrdU staining pretreatment before adding HCl to
> broken DNA helix (brdu can be taken by cell and synthsized into DNA).
> It seems that citrate buffer has more function than simply the antigen
> retrieval.
> 
> Can anyone comment on this further?
> 
> 
> 2008-07-14 
> 
> 
> 
> tf 
> 
> 
> 
> ·¢¼þÈË£º Neil Fournier
> ·¢ËÍÊ±¼ä£º 2008-07-14  03:26:20
> ÊÕ¼þÈË£º tifei <@t> foxmail.com
> ³ËÍ£º 
> Ö÷Ìâ£º [Histonet] Double immunostaining with two same source 1st Antibody
>  
> I am extremely interested in the suggestions you receive. I would like to do a
> similar procedure. If possible, would you be able forward them to me.
> 
> Much appreciated,
> 
> Neil
> 
> Neil M. Fournier
> Department of Psychology
> University of Saskatchewan
> 9 Campus Drive
> Saskatoon, SK, Canada
> S7N 5A5
> 
> Phone:  306-966-4024
>     Fax:  306-966-6630
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet

------------------------------

Message: 7
Date: Mon, 14 Jul 2008 10:11:10 +0800
From: "tf" <tifei <@t> foxmail.com>
Subject: Re: Re: RE: [Histonet] Anterograde tracing
To: "histonet" <histonet <@t> lists.utsouthwestern.edu>
Message-ID: <200807141011050118208 <@t> foxmail.com>
Content-Type: text/plain;	charset="gb2312"

Hi all again.
Just wonder anyone tried WGA-HRP?

it seems that WGA-hRP has a good diffusion ability,, making it ideal for local pressure injection (quite simple method), which PHA-L does bad.
and you can detect it well.

anyone have worked with this?

thx 

2008-07-14 

tf 

·¢¼þÈË£º tf 
·¢ËÍÊ±¼ä£º 2008-07-12  00:50:29 
ÊÕ¼þÈË£º Rachel, Rivka (NIH/NEI) [E]; histonet 
³ËÍ£º 
Ö÷Ìâ£º Re: RE: [Histonet] Anterograde tracing 

Dear Rachel:
Surely I have considered that.
I am actually working on regeneration.
Also, dyes are versatile and go everywhere as long as membrane permits. It does not have the restricted labeling of of one batch of projection and may diffuse a lot when tracing the transection site. Another problem you have mentioned is that DiI does not diffuse well in adult tissue, and thus used more in developmental studies.
thanks a lot
2008-07-12 
tf 
·¢¼þÈË£º Rachel, Rivka (NIH/NEI) [E] 
·¢ËÍÊ±¼ä£º 2008-07-12  00:00:49 
ÊÕ¼þÈË£º tifei <@t> foxmail.com; histonet 
³ËÍ£º 
Ö÷Ìâ£º RE: [Histonet] Anterograde tracing 

What type of tissue are you trying to trace?  If fixed, immature rodent tissue, have you considered one of the fluorescent tracers such as DiI, DiO, DiD, etc. available through Molecular Probes (Invitrogen)?  DiI (red fluorescence) is the brightest.  These dyes don't work well in adult tissue, however.
Rivka
Rivka A. Rachel, MD, PhD
Staff Scientist, National Eye Institute
Neurobiology-Neurodegeneration and Repair Laboratory
Tel: 301 443-4906
-----Original Message-----
From: tf [mailto:tifei <@t> foxmail.com]
Sent: Fri 7/11/2008 11:41 AM
To: histonet
Subject: [Histonet] Anterograde tracing
I just want to discuss the best anterograde tracer.
BDA, PHA-L, CTB-FITC, CTB-Rhodamine, WGA-HRP, HRP, and viral vectors all could be anterograde tracer.
However, BDA is diffusible like all fluroscent dyes, does not have the necessary direction.
CTB-conjugated sereis have limited diffusion ability when injected with pressure, just like PHA-L, thus are not good in labeling a larger group of neurons when single rather multiple injections is required.
WGA-HRP & HRP both antero & retrogradely transported, HRP & virus even transynaptically.
So, any one has other ideas? Such as shorten the period after injection to reduce the diffusion, or using immunodetection to visualize the CTB-FITC, for example?
2008-07-11 
tf 
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Message: 8
Date: Mon, 14 Jul 2008 14:52:26 +0100
From: "Aine Behan" <abehan <@t> rcsi.ie>
Subject: [Histonet] RE: freezing artefact holes on edge of cortex in
	mouse	brain sections
To: <histonet <@t> lists.utsouthwestern.edu>
Message-ID:
	<3DBACD0C44A1CF429431C0C6DC3E81690324DA08 <@t> crc_exchange.rcsi-internal.ie>

Content-Type: text/plain;	charset="iso-8859-1"

Hi all,

I was looking for info on troubleshooting what I think is freezing artefact
holes (FA) in our tissue sections. It is quite specific to the edge of the
brain sections predominantly around the most superficial layers of the
cortex and most obvious in the most lateral aspects of the cortex in coronal
sections.

Brains were fixed in 4% PFA following cervical dislocation and dissection.
They were fixed in graded sucrose concentrations of 10%, 20% and then 30%
over 3 days followed by rapid freezing in isopentane and dry ice. These
brains are stored at -80 until cryostating coronal sections (10 microns
thick). The deeper brain structures are perfectly intact and free from FA
holes.

Has anyone observed this before and do you think this is FA or something
else? I can send on a H&E pic if it proves helpful. It might be a bizarre
question but could there be anything to do with the cryostat that could be
causing this!?!

Regards,

Áine

Áine Behan, PhD

Department of Psychiatry,
Royal College of Surgeons In Ireland,
Smurfit Building,
Beaumont Hospital, 
Dublin 9. 

Ph: +353-1-809-3857/3798 
Fx: +353-1-809-3741

------------------------------

Message: 9
Date: Mon, 14 Jul 2008 22:36:46 +0800
From: "tf" <tifei <@t> foxmail.com>
Subject: Re: [Histonet] RE: freezing artefact holes on edge of cortex
	in	mousebrain sections
To: "Aine Behan" <abehan <@t> rcsi.ie>,	"histonet <@t> lists.utsouthwestern.ed"
	<histonet <@t> lists.utsouthwestern.edu>
Message-ID: <200807142236417873896 <@t> foxmail.com>
Content-Type: text/plain;	charset="gb2312"

Dear Aine:

Yes that's a common problem for cryostat sections.

Different people in my lab has quite diverse response to this.

Some suggested that you can leave the brain in O.C.T before put them in to freezer for 3-6 hours, and the O.C.T will "enter" the brain tissue. Then no "cheese".

Some other people tried the other half of the brain on microtome, to see if this helps. To our surprise, these are no such hollow cavities in microtome-made sections. Thus possibly it's related to O.C.T embedding rather bad dehydration/fixation/post-fixation.

Cheers,

Ti Fei.

2008-07-14 

Department of ANATOMY
The UNIVERSITY OF HONG KONG
LI KAI SHING FACULTY OF MEDICINE
21 Sassoon Road, Pok Fu Lam
Hong Kong

·¢¼þÈË£º Aine Behan 
·¢ËÍÊ±¼ä£º 2008-07-14  21:58:34 
ÊÕ¼þÈË£º histonet <@t> lists.utsouthwestern.edu 
³ËÍ£º 
Ö÷Ìâ£º [Histonet] RE: freezing artefact holes on edge of cortex in mousebrain sections 

Hi all,

I was looking for info on troubleshooting what I think is freezing artefact
holes (FA) in our tissue sections. It is quite specific to the edge of the
brain sections predominantly around the most superficial layers of the
cortex and most obvious in the most lateral aspects of the cortex in coronal
sections.

Brains were fixed in 4% PFA following cervical dislocation and dissection.
They were fixed in graded sucrose concentrations of 10%, 20% and then 30%
over 3 days followed by rapid freezing in isopentane and dry ice. These
brains are stored at -80 until cryostating coronal sections (10 microns
thick). The deeper brain structures are perfectly intact and free from FA
holes.

Has anyone observed this before and do you think this is FA or something
else? I can send on a H&E pic if it proves helpful. It might be a bizarre
question but could there be anything to do with the cryostat that could be
causing this!?!

Regards,

Áine

Áine Behan, PhD
Department of Psychiatry,
Royal College of Surgeons In Ireland,
Smurfit Building,
Beaumont Hospital, 
Dublin 9. 
Ph: +353-1-809-3857/3798 
Fx: +353-1-809-3741

_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu

------------------------------

Message: 10
Date: Mon, 14 Jul 2008 22:40:16 +0800
From: "tf" <tifei <@t> foxmail.com>
Subject: Re: RE: [Histonet] Double immunostaining with two same source
	1st	Antibody
To: "Anatoli Gleiberman" <AGleiberman <@t> cbiolabs.com>
Cc: histonet <histonet <@t> lists.utsouthwestern.edu>
Message-ID: <200807142240108583751 <@t> foxmail.com>
Content-Type: text/plain;	charset="gb2312"

Dear Anatoli:

Thanks very much for your suggestions.

Yes I am using Abcam rat monoclonal Brdu antibody, and I also have tried mouse monoclonal.

SOme people in my lab suggested that without HCl, you may get better DAPI co-staining after BrdU staining, however sometimes you can not get BrdU staining...
We dont know why. Citrate buffer might be able to break some DNA helix during heating process (30 min with 95 degree). But I was just following the protocol.

Next time I will try it. - -

cheers.

2008-07-14 

tf 

·¢¼þÈË£º Anatoli Gleiberman 
·¢ËÍÊ±¼ä£º 2008-07-14  21:57:39 
ÊÕ¼þÈË£º tifei <@t> foxmail.com 
³ËÍ£º 
Ö÷Ìâ£º RE: [Histonet] Double immunostaining with two same source 1st Antibody 

For BrdU staining boiling in citrate buffer is good enough to denature DNA - you don't need to treat sections  with HCl after that. I did it on both paraffin and cryo sections with the same good results. Another suggestion - for BrdU staining use rat monoclonal against BrdU from Apcam - they are much more stronger and cleaner than any mouse monoclonal I have used so far.
Anatoli Gleiberman, PhD
Director of Histopathology
Cleveland Biolabs, Inc
73 High Street
Buffalo, NY 14203
phone:716-849-6810 ext.354
fax:716-849-6817
e-mail: AGleiberman <@t> cbiolabs
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of tf
Sent: Sunday, July 13, 2008 10:02 PM
To: Neil Fournier; histonet
Subject: Re: [Histonet] Double immunostaining with two same source 1st Antibody
Some one suggested a "citrate buffer" treatment before 2nd IHC.
However he als suggested be careful about losing anti-gen in this step.
Actually I used this step for BrdU staining pretreatment before adding HCl to broken DNA helix (brdu can be taken by cell and synthsized into DNA).
It seems that citrate buffer has more function than simply the antigen retrieval.
Can anyone comment on this further?
2008-07-14 
tf 
·¢¼þÈË£º Neil Fournier 
·¢ËÍÊ±¼ä£º 2008-07-14  03:26:20 
ÊÕ¼þÈË£º tifei <@t> foxmail.com 
³ËÍ£º 
Ö÷Ìâ£º [Histonet] Double immunostaining with two same source 1st Antibody 

I am extremely interested in the suggestions you receive. I would like to do a similar procedure. If possible, would you be able forward them to me.
Much appreciated, 
Neil
Neil M. Fournier
Department of Psychology
University of Saskatchewan
9 Campus Drive
Saskatoon, SK, Canada
S7N 5A5
Phone:  306-966-4024
    Fax:  306-966-6630 

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Message: 11
Date: Mon, 14 Jul 2008 11:07:12 -0400
From: "Blazek, Linda" <lblazek <@t> digestivespecialists.com>
Subject: [Histonet] Schiff's removal
To: "histonet <@t> pathology.swmed.edu" <histonet <@t> pathology.swmed.edu>
Message-ID:
	<5A2BD13465E061429D6455C8D6B40E390B77E277 <@t> IBMB7Exchange.digestivespecialists.com>

Content-Type: text/plain; charset="us-ascii"

To who ever made the suggestion of using Anatech hand cleaner for Schiff's reagent to decolorize a PAS stain gets a GREAT BIG thanks!  A whole rack of slides inadvertently got stained with the Alcian Blue/PAS.  Anatech's hand cleaner did magic!

Linda Blazek HT (ASCP)
Manager/Supervisor
GI Pathology of Dayton
7415 Brandt Pike
Huber Heights, OH 45424
Phone: (937) 293-4424 ext 7118
Email: <mailto:lblazek <@t> digestivespecialists.com> lblazek <@t> digestivespecialists.com<mailto:lblazek <@t> digestivespecialists.com>
P Please consider the environment before printing this e-mail.

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Message: 12
Date: Mon, 14 Jul 2008 08:57:36 -0700 (PDT)
From: eplurbus <@t> u.washington.edu
Subject: [Histonet] NBT/BCIP staining for Alkaline Phosphatase
To: histonet <@t> lists.utsouthwestern.edu
Message-ID:
	<Pine.LNX.4.43.0807140857360.12299 <@t> hymn34.u.washington.edu>
Content-Type: TEXT/PLAIN; charset=US-ASCII; format=flowed

Hello all,

I am condicting a bone histomorphometry study that involves staining for detection of alkaline phosphatase on methymethacrylate embedded sections. I am able to get OK results with Roche brand NBT/BCIP ready-to-use tablets but I was wondering if anyone out there would recommend another specific brand of tablets that they have had good success with.

Also, I am currently using an old bottle of Zymed Clearmount to mount the cover slips. I am not so pleased with the results. Can anyone recommend a good water based mountant?

Thanks

Alexander Dowell
University of Washington
eplurbus <@t> u.washington.edu

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End of Histonet Digest, Vol 56, Issue 17
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